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Paroxysmal Nocturnal Hemoglobinuria - PNH

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, potentially life-threatening acquired stem cell disorder caused by a variant in the PIGA gene. The variant leads to a lack of glycosylphosphatidylinositol (GPI)-anchored proteins on the surface of blood cells, which in turn leads to an inappropriate immune response to, and hemolysis of, these cells. In addition to hemolysis, PNH is characterized by thrombosis and bone marrow failure. Although some patients may present with the nocturnal hemoglobinuria for which the disease is named, the common symptoms of PNH are nonspecific (eg, fatigue, dyspnea, anemia, abdominal pain), which presents a diagnostic challenge. A delayed or missed diagnosis prevents patients from receiving appropriate treatment and thereby decreases quality of life and impacts survival. Laboratory testing in PNH includes flow cytometry to diagnose PNH in patients with suggestive symptoms or test results. Laboratory testing is also used to classify PNH in order to inform treatment and monitor disease progression and the effects of treatment.

Quick Answers for Clinicians

Which is the first-line test in patients with suspected paroxysmal nocturnal hemoglobinuria?

When a patient has been assessed for hemolysis (see Initial Evaluation) and paroxysmal nocturnal hemoglobinuria (PNH) is suspected, the recommended test is flow cytometry to evaluate for the presence of glycosylphosphatidylinositol (GPI)-linked antigens on blood cells. Both red blood cells (RBCs) and white blood cells (WBCs) should be analyzed. For additional details, see Flow Cytometry.

What is the pathophysiology of paroxysmal nocturnal hemoglobinuria?

Paroxysmal nocturnal hemoglobinuria (PNH) arises due to variants in the PIGA gene, which result in the deficiency or absence of glycosylphosphatidylinositol (GPI)-anchored cell membrane proteins. This in turn leads to a partial or complete deficiency of CD55 and CD59, two important molecules in the complement cascade, on the surface of hematopoietic stem cells. These stem cells clonally expand into red blood cells (RBCs) and white blood cells (WBCs) that are susceptible to lysis by complement. The resulting hemolysis leads to a number of symptoms; however, the pathophysiology of the bone marrow failure and thromboses often observed in PNH is unknown.

Should the Ham test and sugar water test be used in the diagnosis of paroxysmal nocturnal hemoglobinuria?

The Ham test (acid hemolysin) and the sugar water test (sucrose hemolysis) are no longer routinely used for the assessment of paroxysmal nocturnal hemoglobinuria (PNH) due to their lack of specificity, less quantitative nature, and difficulty.

Indications for Testing

Laboratory testing for PNH is appropriate in patients with the following clinical indications:

Hemoglobinuria
Unexplained hemolysis with iron deficiency, abdominal pain, thrombosis, dysphagia, or granulocytopenia/thrombocytopenia
Coombs-negative hemolytic anemia that cannot be otherwise explained
Thrombosis at unusual sites or with unexplained hemolytic anemia or cytopenia
Evidence of bone marrow failure (aplastic anemia, hypoplastic anemia, myelodysplastic syndrome)

Testing can also be used to monitor disease progression and response to treatment in patients with confirmed PNH.

Laboratory Testing

Initial Evaluation

A CBC with peripheral smear, reticulocyte count, and Coombs test should be performed in all cases of suspected PNH to assess anemia. Depending on whether the patient has classic PNH or PNH in the context of another disorder (see Classification of PNH table), leukocyte and platelet counts may be normal or low. A reticulocyte count is also recommended to assess the bone marrow response to anemia. Reticulocyte counts are generally elevated in PNH, but are lower than would be expected given the severity of anemia. A Coombs test should be performed to rule out other causes of anemia, given that PNH is Coombs negative.

Serum lactate dehydrogenase (LDH), indirect bilirubin, and serum haptoglobin tests are useful for assessing hemolysis. Serum LDH is nearly always elevated in clinical PNH, although the level of elevation depends on the PNH classification. Indirect bilirubin may be elevated, and serum haptoglobin is generally low, although it should be noted that patients with subclinical PNH will not exhibit any evidence of hemolysis.

Diagnosis

Flow Cytometry

Flow cytometry is the preferred technique for the diagnosis of PNH and is performed to evaluate for the presence of GPI-linked antigens on blood cells. Peripheral blood is the preferred specimen, and the use of multiple specific GPI-linked reagents is recommended. Flow cytometry enables calculation of the percentage of red blood cells (RBCs) or white blood cells (WBCs) that entirely or partially lack GPI-linked antigens compared with normal cells; this percentage is referred to as the PNH clone size.

Determination of both WBC and RBC clone size is recommended for diagnosis because there are limitations associated with either WBC or RBC testing alone. WBC analysis using fluorescently labeled aerolysin (FLAER) and CD157 as GPI-linked markers, as well as CD15 and CD64 as lineage-specific markers for granulocytes and monocytes, respectively, is the most accurate test for PNH clone size. Analysis of RBCs alone may lead to an underestimate of the overall PNH clone size because a patient may have received a transfusion of normal RBCs. WBC testing, however, is less useful for the detection of cells that only partially lack GPI-linked antigens.

Other Tests

Testing for a PIGA gene variant can be used to confirm a diagnosis of PNH; however, this testing is not widely performed. Other tests for GPI-anchored proteins, such as erythrocyte acetylcholine esterase or neutrophil alkaline phosphatase assays, can be used to support a diagnosis made via flow cytometry but are not recommended for standalone use.

Classification

Once clone size has been determined, PNH can be classified according to presentation, which enables the development of an appropriate treatment plan.

Classification of PNH
Classification	Presentation	Markers	PNH Clone Size
Classic PNH	Hemolysis and/or thrombosis	Reticulocytosis, high LDH, high bilirubin, low haptoglobin, normal leukocyte and platelet counts	Large (>50%)
PNH in the context of another disorder	Primary bone marrow disorder (eg, aplastic anemia, myelodysplastic syndrome)	Reticulocytosis, variable LDH, high bilirubin, low haptoglobin, low leukocyte and platelet counts	Varies, but generally small (<50%)
Subclinical PNH	No hemolysis or thrombosis	Normal or near normal LDH, bilirubin, and haptoglobin	Small (<10%)
Sources: Parker, 2016; Borowitz, 2010; Parker, 2005

Bone Marrow Examination

Histologic examination of bone marrow is required to identify primary bone marrow disorders and thus classify PNH. Bone marrow examination is indicated when bone marrow transplantation is being considered and to rule out other disorders when pancytopenia is present. Cytogenetic analysis of bone marrow samples is recommended to help identify underlying disease processes associated with PNH.

Monitoring

Flow Cytometry

Regular monitoring of clone size with flow cytometry is recommended in patients diagnosed with PNH to assess disease progression. Annual monitoring is recommended for patients with stable PNH; more frequent monitoring is suggested if the clone size is changing. Monitoring is also recommended to assess response to therapy, if the patient is receiving eculizumab, and to determine transfusion needs.

WBC analysis is the most reliable test for PNH clone size (as mentioned above), but RBC analysis is the most appropriate test to monitor subclinical PNH and response to eculizumab treatment, particularly during stabilization of the RBC clone. RBCs also remain suitable for testing longer than WBCs, and RBC testing can be used to quantify cells that are only partially deficient in GPI-anchored proteins.

Other Tests

Serum LDH generally decreases to near normal levels in patients treated with eculizumab. However, laboratory evidence of anemia and hemolysis may persist, regardless of the success of treatment. Iron stores and serum erythropoietin concentrations should be examined in patients being treated with eculizumab who have persistent anemia to determine whether additional treatment to facilitate erythropoiesis is warranted.